E. coli trp operon

Transcription Attenuation How would transcription of the E. coli trp operon be affected by the following manipulations of the leader region of the trp mRNA ... 

TrpR, which is a DNA binding repressor protein, regulates transcription initiation of the E. coli trpEDCBA operon. Under tryptophan limiting conditions, TrpR represses transcription initiation, whereas repression is relieved in the presence of excess tryptophan. Once transcription initiates the elongating transcription complex is subject to control by transcription attenuation (reviewed in References 5 and 6). The leader transcript can form three RNA secondary structures that are referred to as the pause hairpin, the antiterminator structure, and an intrinsic terminator hairpin. Because the antiterminator shares nucleotides in common with the terminator, their formation is mutually exclusive. The pause hairpin has two additional roles in this transcription attenuation mechanism it serves as an anti-antiterminator stmc-ture that prevents antiterminator formation, and it codes for a leader peptide. A model of the E. coli trp operon transcription attenuation mechanism is presented in Fig. 2a. 

Base sequence of (a) the DNA of the E. coli trp operon at which transcription termination occurs and of (b) the y terminus of the mRNA molecule. The inverted-repeat sequence is indicated by reversed arrows. The mRNA molecule is folded to form a stem-and-loop structure. 

Attenuation. A major mechanism of feedback repression, known as attenuation, depends not upon a repressor protein but upon control of premature termination. It was first worked out in detail by Yanofsky et al. for the trp operon of E. coli and related bacteria.184 186 Accumulation of tryptophan in the cell represses the trp biosynthetic operon by the action of accumulating tryptophanyl-tRNATlP, which specifically induces termination in the trp operon. Other specific "charged" arnino-acyl-tRNA molecules induce termination at other amino acid synthesis operons. 

Figure 13.2 E. coli tryptophan (trp) operon. o, operator L, leader a, attenuator E, D, C, B, A, structural genes. Numbers are base pairs.

Figure 29.4 Prokaryotic promoter sequences. A comparison of five sequences from prokaryotic promoters reveals a recurring sequence of TATAAT centered on position —10. The 10 consensus sequence (in red) was deduced from a large number of promoter sequences. The sequences are from the (A) lac, (B) gal, and (C) trp operons of . coli from (D) X phage and from (E) ( )X174 phage.

In E. coli, about half the genes are clustered into oper-ons each of which encodes enzymes involved in a particular metabolic pathway or proteins that interact to form one multisubunit protein. For instance, the trp operon mentioned earlier encodes five enzymes needed in the biosynthesis of tryptophan (see Figure 4-12). Similarly, the lac operon encodes three enzymes required for the metabolism of lactose, a sugar present in milk. Since a bacterial operon is tran-... 

The E. coli tryptophan (trp) operon (Fig. 28-19) includes five genes for the enzymes required to convert chorismate to tryptophan. Note that two of the enzymes catalyze more than one step in the pathway. The mRNA from the trp operon has a half-life of only about 3 min, allowing the cell to respond rapidly to changing needs for this amino acid. The Trp repressor is a homodimer, each subunit containing 107 amino acid residues (Fig. 

Fig. 1. Attenuator site in the leader of the tryptophan synthesis operon of E. coli, and the compiete sequence of the terminated leader RNA. Two regions in the trp attenuator DNA have a 2-fold axis of symmetry. A, B, C. D in the RNA correspond to similarly labeled regions in Fig. 2. Base pairing with resulting stem and loop formation is possible between A and B (free energy of formation -46.9 kJ/mol), B and C (-49 kJ/mol), C and D (-83.7 kJ/mol). (lodons for the leader peptide are also shown and the two strategic Tip codons are indicated by asterisks.

The first structural gene of the operon is separated from the promoter-operator by a length of DNA called the leader. Transcription of the operon proceeds via this leader and into the structural genes. For example, in the case of the trp operon of E. coli, a single continuous 7,000 nucleotide trp mRNA transcript is formed, which includes leader RNA (162 nucleotides) at the S end, followed by mRNA sequences for all the enzymes in the biosynthetic pathway. This transcript is formed only when tryptophan is re-... 

Negative repression was investigated in the tryptophan (trp) synthesis operons of E. coli and Salmonella typhimurium, and the histidine synthesis operon of Salmonella. The trp operon is totally derepressed in the presence of nonsurplus amounts of tryptophan. Here, transcription and translation go at maximal rates. Enzymes of the tryptophan system synthesized it in amounts which are totally utilized during translation. This condition is known as turn on. The activity of the first enzyme in the sequence of tryptophan synthesis — anthranilate synthetase — is inhibited within several seconds after the addition of surplus tryptophan. The aporepressor becomes the repressor and inhibits transcription of the operon (negative repression) after it associates with the effector, tryptophan. This association occurs if the surplus of tryptophan exists for a sufficient amount of time. After several minutes, most of the resulting mRNA is degraded and the rate of synthesis is noticeably reduced (known as the turn of condition). 

E. coli promoters are those from the lac or trp operons and the late promoter, pL, from phage lambda. These promoters can be turned on by the addition of an inducer (derepressor) such as IPTG (isopropyl thiogalactoside) or lactose for lac, or lAA (indoleacrylic acid) for trp, or by a temperature shift from 30 to 37°C to 42°C for pL. Following process optimization and scale-up, an expression level of 1 to 5 g/liter is achievable with E. coli expression cultures. 

Induction refers to the stimulation of the synthesis of a catabolic enzyme, which is not currently being made by the cell, by the presence of its substrate or a substrate analogue (a substance closely related to the substrate). Repression refers to the inhibition of the synthesis of an enzyme involved in an anabolic pathway when its reaction product reaches a sufficiently high concentration. The two classical examples of induction and repression in E. coli are the lac operon and the trp operon respectively. 

FIGURE 17.10 The trp operon of E. coli. (a) Organization of the trp operon showing bound RNA polymerase, (b) Inhibition of enzyme synthesis in the presence of tryptophan... 

Morse, D. E., Mosteller, R. D., Yanofsky, C. Dynamics of synthesis, translation, and degradation of trp operon messenger RNA in E. coli. Cold Spr. Harb. Symp. quant. Biol. 34, 725-740 (1969). 

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